Chlorinated hydrocarbons represent a class of toxic chemicals frequently found at contaminated sites. Perchloroethene (e.g., tetrachloroethylene “PCE”) and trichloroethene (“TCE”) are choice solvents for numerous applications, but their widespread use has resulted in extensive groundwater contamination. Partial reductive dechlorination of PCE and TCE mediated through abiotic and biotic processes lead to the accumulation of toxic (e.g., dichloroethenes, “DCEs”) and carcinogenic (e.g., vinyl chloride, “VC”) intermediates. See, e.g., Campbell et al., Environ. Toxicol. Chem., 1977, 16: 625-630; Allen-King, et al., Environ. Toxicol. Chem., 1997, 16:424-429; Abelson, Science, 1990, 250:733; DiStefano, et al., Appl. Environ. Microbiol., 1991, 57:2287-2292; Freedman, et al. Appl. Environ. Microbiol., 1989, 55:2144-2151; Vogel, et al., Appl. Environ. Microbiol., 1985, 49:1080-1083; Maymó-Gatell, et al., Appl. Environ. Microbiol., 1995, 61:3928-3933. VC has been found in at least 496 of the 1,430 National Priorities List (NPL) sites identified by the U.S. Environmental Protection Agency (EPA). PCE and TCE are present in at least 771 and 852 NPL sites, respectively. See, e.g., EPA, Agency for Toxic Substances and Disease Registry, ToxFAQs for chlorinated ethenes. 1996; on the Worldwide Web at www.atsdr.cdc.gov/tfacts70.html.
Although the main contributions of VC contamination in subsurface environments is through the incomplete reductive dechlorination of PCE and TCE, other contamination sources exist. For example, past incidental releases of VC may have led to local groundwater and soil contamination. For instance, VC is a precursor in the manufacturing of PVC, and in excess of 7 million tons of VC was produced in the United States in 1996. In recent years, the estimated annual VC production in the world was 27 million tons. See, Kielhorn, et al., Environ. Health Perspect., 2000, 108:579-588. PVC is also a suspected source of VC in landfills, where VC is frequently detected in drainage water. See, Coulston, et al., Regul. Toxicol. Pharmocol., 1994, 19:344-348.
The remediation of groundwater contaminated with chlorinated ethenes is challenging. Traditional “pump-and-treat” treatment systems have proven to be ineffective, time-consuming and costly, especially at contaminated sites with complex hydrogeology and large plumes. The discovery that bacterial populations use chlorinated ethenes as electron acceptors, thus efficiently reducing and detoxifying these compounds, has made bioremediation an attractive technique for ground water pollution control. This process, in which bacteria couple the reductive dechlorination process to growth, is known as (de)chlororespiration or chloridogenesis. See e.g., Löffler et al., Appl. Environ. Microbiol., 1996, 62:3809-3813. The physiology and phylogeny of several PCE-to-cis-DCE-dechlorinating bacteria are fairly well understood and have received ample review. See e.g., Holliger et al., FEMS Microbiol. Rev. 1999, 22:383-398.
Although the complete microbial reductive dechlorination of chloroethenes to ethene is well documented in microcosms, laboratory cultures, and bioreactors, the nature of the organisms responsible for the final dechlorination step remains elusive. Both Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain FL2 were shown to reduce VC to ethene, however, the reaction was cometabolic, only occurring when the cultures were grown with a higher chlorinated ethene. Neither population grew with VC alone. See, e.g., Löffler et al., Appl. Environ. Microbiol., 2000, 66:1369-1374. Flynn et al. demonstrated a community shift in response to enrichment with PCE versus cis-DCE or VC. Flynn et al., Environ. Sci. Technol., 2000, 34:1056-1061. Circumstantial evidence strongly suggested that populations that use VC as a metabolic electron acceptor exist. Löffler et al., Appl. Environ. Microbiol., 1999 65:4049-4056; Rosner et al., Appl. Environ. Microbiol., 1997, 63:4139-4144.
Hendrickson et al. detected Dehalococcoides 16S rRNA sequences at 21 chloroethene-contaminated sites that produced ethene. Hendrickson et al., Appl. Environ. Microbiol., 2002, 68:485-495. Another study demonstrated that five enrichment cultures that were maintained with VC as electron acceptor, dechlorinated VC to ethene in the absence of polychlorinated ethenes. All five cultures contained at least one Dehalococcoides population, as demonstrated with 16S rRNA gene-based approaches. K. M. Ritalahti et al., Abstr. 6th Int. Symp. In situ On-Site Bioremediation, 2001. These findings imply that members of the Dehalococcoides cluster with different properties than the PCE/TCE-dechlorinating isolates Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain FL2 are involved in metabolic VC reductive dechlorination.
It is desired to characterize a VC-to-ethene-dechlorinating enrichment culture obtained from an impacted aquifer or other contamination site, to identify the population(s) catalyzing the dechlorination step, and to demonstrate that VC serves as a growth-supporting electron accepter for the dechlorinating population(s).
This goal, as well as optimal functioning of this bioremediation technique in general, depends on precise quantification of Dehalococcoides species, like those discussed above. Thus to there is a need for accurate quantitative analysis of Dehalococcoides populations. Such measurements are preferably precise and obtained quickly, thus producing an accurate assessment of Dehalococcoides species while reducing lag time.
Previously, PCR-based efforts to quantify Dehalococcoides species have employed 5′AAGGCGGTTTTCTAGGTTGTCAC3′ (SEQ ID NO: 6) as the forward primer, and 5′CGTTTCGCGGGGCAGTCT3′ (SEQ ID NO: 7) as the reverse primer. Löffler, Appl. Environ. Microbiol., 2000, 66:1369-1374. International Publication Number WO 00/63443 sets forth various sequences that can be used as primers and probes said to be useful in the identification of dechlorinating bacteria. These primers were derived from specific segments of the 16S rRNA gene of Dehalococcoides ethenogenes (DHE).
Notwithstanding the publication of certain primer sets for PCR amplification of Dehalococcoides 16S RNA genes, there remains a need in the art for an efficient real time detection system, particularly one that works in the field. There also is a need for obtaining template DNA for the Dehalococcoides species of interest that is suitable for PCR amplification.
In the present application, the inventors describe primers and probes adapted for Real Time PCR, and provide enhanced specificity for Dehalococcoides, and are different from the primer pairs and probes known in the art, and methods for preparing samples for RTm-PCR detection of Dehalococcoides. 